Evaluation of the Efficiency of Some Antagonistic Trichoderma spp. in the Management of Plant Parasitic Nematodes

Plant parasitic nematodes cause great economic losses to agricultural crops worldwide. They along, with their hosts, are not isolated in the ecological system, but are strongly influenced by antagonists, parasites and pathogens. Though pesticides appear to be the most economical and efficacious means of controlling plant pathogens, toxicological, environmental and sociological concerns have led to drastic reductions in the availabil‐ ity of efficient commercial nematicides. These restrictions have forced farmers to look for an integral system that makes use of other means of disease control. Species of spiral nematodes, Helicotylenchus and Scutellonema , were among the most abundant plant parasitic nematodes of the mulberry plant. Eco-friendly control of the parasitic nematodes could be achieved by means of endoparasitic fungi (like Hirsutella , Meria , Nematophthora and Nematoctonus ), trapping fungi (like Arthrobotrys and Duddingtonia ) or parasitic fungi (like Paeceilomyces lilacinus ). During the course of this present work, Trichoderma Pers. Ex. Fr. was found to be one of the most effective fungi in controlling the eggs and J 2 of Meloidogyne javanica . The present study outlines the comparative efficacy of five Trichoderma species ( T. viride, T. harzianum, T. longibrachiatum, T. koningii and T. hamatum ) against Helicotylenchus sp. and Scutellonema sp. The study also outlines the effect of Trichoderma viride Persoon on Scutellonema spp. and Helicotylenchus sp., effect of Trichoderma harzianum Raifae on Scutellonema sp. and Helicotylenchus sp., effect of Trichoderma longibrachiatum Rifai on Scutellonema sp. and Helicotylenchus sp., effect of Trichoderma koningii Oudeom on Scutellonema sp. and Helicotylenchus sp., and lastly effect of Trichoderma hamatum (Bonord) Bainier on Scutellonema sp. and Helicotylenchus sp.


Introduction
Plant parasitic nematodes cause great economic losses to agricultural crops worldwide. They along, with their hosts, are not isolated in the ecological system, but are strongly influenced by antagonists, parasites and pathogens. Though pesticides appear to be the most economical and efficacious means of controlling plant pathogens, toxicological, environmental and sociological concerns have led to drastic reductions in the availability of efficient commercial nematicides. These restrictions have forced farmers to look for an integral system that makes use of other means of disease control. This imperative approach involves a mixture of agrotechnical, biological, chemical and genetic (breeding) means of plant disease control [20,24,36]. Species of spiral nematodes, Helicotylenchus and Scutellonema, were among the most abundant plant parasitic nematodes of the mulberry plant. Reductions in length and weight of shoot, number and weight of leaves, and number of leaf buds were the characteristic symptoms of the infection of spiral nematodes [10]. Rao and Swarup [26] found stunting of the plants and reduction in fresh and dry weights of both shoot and root system in sugarcane due to Helicotylenchus dihystera. Besides chemicals, various researchers suggested other control measures in view of the need to replace highly toxic and potentially polluting chemicals used to control plant parasitic nematodes and fungi with less dangerous chemicals, or preferably with biological control agents and botanicals [21]). The discovery of new biocontrol agents and the demonstration of their value in reducing disease incidence and severity has opened promising new avenues for practical applications in agriculture as well as for promoting environmental safety [8]. Considerable efforts have been made by many researchers for the management of different plant parasitic nematodes with the use of Trichoderma harzianum [1 -5, 23, 28, 33].
Eco-friendly control of the parasitic nematodes could be achieved by means of endoparasitic fungi (like Hirsutella, Meria, Nematophthora and Nematoctonus), trapping fungi (like Arthrobotrys and Duddingtonia) or parasitic fungi (like Paeceilomyces lilacinus). But there are problems in the culture of the fungi, such as unavailability of their host, and the generalist feeding nature of fungi that means they can become trapped on and digest beneficial as well as pest species of nematode. The general approach has been to go to locations where nematodes have reached high densities, and extract parasitized individuals from the soil. Then, the fungi were cultured and tested as parasites of the nematode pest. The mycoparasitic ability of Trichoderma sp. against soil-borne plant pathogens allows for the development of biocontrol strategies [11,13,14,16,24]). Windham et al. [40] reported reduced egg production in the root-knot nematode Meloidogyne arenaria following soil treatment with Trichoderma harzianum and T. koningii preparations. Combining T. harzianum with neem cakes reduced the population of citrus nematode, Tylenchulus semipenetrans [25]. Reduction of M. javanica infection with several isolates of Trichoderma lingnorum and T. harzianum has been reported [32]. Trichoderma may also promote plant growth [19].
During the course of this present work, Trichoderma Pers. Ex. Fr. was found to be one of the most effective fungi in controlling the eggs and J 2 of Meloidogyne javanica. The fungi is characterized by rapidly growing colonies bearing tufted or postulate, repeatedly branched coni-diophores with lageniform phialides and hyaline or green conidia borne in slimy heads. They can be cultured and isolated from any type of soil. Considering the importance of the fungal genus containing species that have the potential for economic impact, the present study was carried out to determine the comparative efficacy of five Trichoderma species (T. viride, T. harzianum, T. longibrachiatum, T. koningii and T. hamatum) against Helicotylenchus sp. and Scutellonema sp.

Extraction of nematodes
Soil samples from around the rhizospheric regions of mulberry plants were collected and processed through Cobb's sieving and decanting method followed by Baerman's funnel technique [38]. The nematodes were observed under stereoscopic microscope and were counted using a Syracuse counting disc.

Isolation and enumeration of Trichoderma sp. from soil
The fungi were isolated through the serial soil dilution plate method [39]. Then, 10 g of oven dried fungi was added to a sterile Erlenmeyer flask with 90 ml sterile water, and the mixture was stirred with a magnetic stirrer for 20-30 minutes. A blender was used for blending the samples. While the suspension was in motion, 10 ml of solution was taken and added to 90 ml sterile water in a screw-cap flask or medicine bottle. It was shaken for one minute and 10 ml of the suspension was transferred to a 90 ml sterile water blank. The process was repeated until the desired dilution was obtained. Ten millilitres of soil solution was pipetted and mixed with 90 ml distilled water and marked to 10 -3 . From 10 -2 and 10 -3 test tubes about 5 ml solution was added to culture media contained in four petri dishes (two of each) and kept at laminar flow for 3-4 days.
To facilitate uniform spreading of the suspension over Czapek Dox agar surface at pH 5.5, the plate was placed on a turntable and the suspension spread with a flamed L-shaped rod with one hand, while rotating the turntable with the other. To obtain distinct colonies, plates were prepared 2-3 days before use or placed for a few hours at 35 to 40° C after pouring to ensure a dry agar when the suspension was added. A water film on the freshly poured plates caused excessive spreading of organisms. The plate was incubated for a few days at 24-30° C and colony counted. A broth media was made by mixing together the substances excepting the agar. It was kept for 24 hours to dissolve the substances completely. Five to six drops of the broth were removed with a dropper into different autoclaved cavity blocks.

Inoculation of nematode and fungi
Five to six drops of the broth were removed with a dropper into different autoclaved cavity blocks. Then, 0.1 ml of selected Trichoderma spp. was transferred into the cavity block containing the broth. Next, 200 female Scutellonema spp. and Helicotylenchus spp. each were also inoculated into the cavity blocks. The cavity blocks containing the whole mixture were incubated at room temperature covered upside down by autoclaved petri dishes. Uninoculated nematodes on the broth were also kept as control. Observation of the nematodes under stereoscopic binocular microscope to record their mobility and fungal infection was done at each 12-hour interval. Each treatment was replicated three times.

Results and Discussion
The fungus attacked the nematodes though the production of conidia, sticky spores and mycelia, which on contact adhere to the cuticle and germinate, forming germ tubes that penetrated the nematodes. Then, they extended their hyphae inside the nematodes after penetration of the cuticle by conidia formation. These hyphae multiplied profusely. They inactivated the parasitic nematodes and immobilized them due to production of certain antibiotics and compounds. Observations of the immobility and parasitism of the nematodes due to the fungi were made every 12 hours. Each observation was replicated three times and the results are represented in tables 1-10. Photographs with graphs of parasitism are also provided in figures 1-32.

Effect of Trichoderma viride Persoon on Scutellonema spp. and Helicotylenchus sp. (table 1 and 2)
After 12 hours of inoculation, the fungus produced mycelium and conidia. The highest immobility was found at 108 hours of inoculation. The spores attached at the middle and anterior end of the body, and made the nematode immobile in the case of Scutellonema spp. After 24 hours of inoculation, many mycelium were found attached to the entire body of the nematode, due to which the body of the nematode was deformed and became shrunken, killing many of the nematodes. Body constrictions of nematodes might be due to the sucking of body contents by the fungus. Fifty percent of Scutellonema spp. out of 200 nematodes were immo-bilized by the fungus at 84 hours, and complete immobilization was observed at 108 hours of inoculation. In the case of Helicotylenchus spp., infection started within 24 hours of inoculation, and complete infestation occurred within 4-5 days of inoculation. The conidiophores of Trichoderma viride were less complicated; they formed aerial hyphae and coiled around the body of the nematode, producing smaller branches and ultimately forming a conifer-like branching system.

Effect of Trichoderma harzianum Raifae on Scutellonema sp. and Helicotylenchus sp. (table 3 and 4)
There was no infection after 24 hours of inoculation, but 3 % of the nematodes were immobile. Infection and parasitism of the nematode occurred after 48 hours of inoculation. The highest immobility was found at 108 hours of inoculation. Many mycelia grew over the body of the nematode. The conidiophores were seen to be multiple-branched, forming loose tufts which arose in distinct and continuous ring-like zones. The main branches, mostly in groups of two or three, stood at right angles, and the length increased with the distance from the tip of the main branch, giving a conical or pyramidal appearance. The body cuticle of the nematode was suppressed. The mycelia tip ran parallel to the nematode. There was rapid and excessive coiling on the target host. The mycelium coiled with its constricting networks of loops at the anterior region of the body and the head region, making constrictions that might be due to the sucking of body contents. After 96 hours of inoculation, there was complete immobilization of the nematodes. In the case of Helicotylenchus, the highest percentage of infection and immobility occurred during 96 th hour of inoculation.

Effect of Trichoderma longibrachiatum Rifai on Scutellonema sp. and Helicotylenchus sp. (table 5 and 6)
After 12 hours of inoculation, 4 % of the total nematode population was found to be immobile, with the highest immobility at 108 hours of inoculation. Infection started before 20 hours of inoculation. Hyphae of the fungus strain formed an appressorium-like structure in close contact with the nematode. They produced penetration holes in the cuticle of the nematode. The penetrated cuticle rapidly lost turgor and collapsed. At contact with the nematode cuticle, the hyphae branched dichotomously at the tip. The hyphae were not observed to coil around the nematode cuticle, and instead grew along the cuticle. However, penetration was not evident. Despite the absence of visible penetration, the nematode cuticle lost turgor pressure, wrinkled and collapsed. Finally, both the cuticle and body content of the nematode completely disintegrated. In the case of Helicotylenchus, the highest immobility was found at 60 hours of inoculation.    There was no effect during the first 12 hours of inoculation in the case of Helicotylenchus spp., but 7 % of Scutellonema spp. were immobilized during that time. After 24 hours of exposure, conidia attachment of the nematode was found. The conidia stuck towards the cephalic region and stylet of the nematode. Maximum immobility in the case of Scutellonema occurred at 144 hours of nematode exposure to the fungus, while it occurred at 168 hours of exposure in the case of Helicotylenchus sp. At 48 hours of exposure, hyphae formation was found around the body of Helicotylenchus and at the anterior and posterior part of the body of Scutellonema spp. The hyphae penetrated towards the body cuticle of the nematode and sucked the body contents, affecting the nematode. This might be attributed to the fungus's production of endoand exochitinases by which hyphae penetration of the nematode cuticle was made possible.

Effect of Trichoderma hamatum (Bonord) Bainier on Scutellonema sp. and Helicotylenchus sp. (table 9 and 10)
There was no effect on the nematode Scutellonema spp. during the first 60 hours of exposure to the fungus, or 72 hours in the case of Helicotylenchus sp. Immobilizations of a few Scutellonema sp. were found at 72 hours of exposure, while this occurred at 96 hours of exposure in the case of Helicotylenchus sp. Hundred percent immobility of Scutellonema sp. was found at 300 hours of inoculation, and in the case of Helicotylenchus, it was found at 444 hours of exposure. Infection of Scutellonema sp. started after 68 hours of exposure, and 80 hours in case of Helicotylenchus. Direct growths of the mycoparasite from the body of the nematodes were observed. There was spore formation inside the body of the nematode and shrinkage of body contents occurred. Trichoderma hamatum produced aspersoria-like structures attached to the host cell wall. Subsequently several different types of interaction occurred. The fungus either grew parallel to and along the host hyphae or coiled around the host. In Helicotylenchus sp., the parasite penetrated into and grew within the cuticle. The cuticle became vacuolated, shrank, collapsed and finally disintegrated. The oesophageal part of the nematode had shrunken and the tail region was disintegrated into two, as in a fork.    Several possible mechanisms have been suggested to be involved in Trichoderma antagonism, such as production of volatile or non-volatile antibiotics by the fungus [6], space-or nutrient-(carbon, nitrogen, iron, etc.) limiting factors that compete with the host [31], and direct mycoparasitism whereby the host cell wall is degraded by the lytic enzymes secreted by Trichoderma [9]. Trichoderma harzianum produced antibiotic 6-pentyl-α-pyrone, which had the dual effect of inhibiting pathogen growth and down-regulating genes for biosynthesis of trichothecenes, a class of mycotoxins with broad-spectrum antimicrobial activity [12]. Trichoderma longibrachiatum produced three main hydrolytic enzymes: protease, β-1, 3glucanase and chitinase, which were involved in fungal cell wall degradation. Trichoderma koningii has also been found to produce cell wall degrading enzymes -chitinases, β-1, 3glucanase and cellulose -which aid in the colonization of their host cells, while isonitrin, homothalin A, melanoxadin, trichodermin, ergokonin, viridian, viridio fungin A, B and C produced by the fungus act in antibiosis [22]. Sharon et al. [29] studied the mechanism involved in the attachment and parasitic processes with special emphasis on the important role of the nematode's gelatinous matrix (gm) in direct nematode-fungus interactions, and suggested that carbohydrate-lectin-like interactions might be involved in the attachment of conidia to the nematodes. The authors also found that parasitism was one of the modes of action of Trichoderma species against Meloidogyne javanica. Trichoderma longibrachiatum produced nematotoxic concentrations of acetic acid. Secondary metabolites from fungi also contained compounds which were toxic to plant parasitic nematodes [17,30]. Trichoderma are also known to produce toxins and antibiotics like malformin, hadacidine, gliotoxin, viridian and penicillin [37], which might contribute to the inactivity and immobility of the nematodes. Parasitic interactions between Trichoderma and nematodes might take place in soil, on root surfaces [29] and in the rhizosphere, sites that could be colonized by these opportunistic avirulent plant symbionts [18]. The improved attachment and parasitism observed in vitro could facilitate the development of new strategies to affect interactions between the nematode, plant and fungus for successful biocontrol.
The tested species of Trichoderma show a significant effect on the activity of nematodes. The results indicated that T. harzianum followed by T. longibrachiatum, T. viride, T. koningii and T. hamatum were effective in controlling the plant parasitic nematodes Helicotylenchus sp. and Scutellonema sp. Trichoderma sp. was considered imperfect filamentous (Deuetromycetes, Hyphomycetes, Phialasporace, Hyphales, Dematiaceae), and was the most common saprophytic fungi in the rhizosphere found in almost any soil.
Using beneficial fungi for control of plant disease is a useful and acceptable method for farmers. As such, the fungal microbe Trichoderma sp. can be used in biological control in the Integrated Pest Management (IPM) programme to achieve good success. Among the tested species of Trichoderma, Trichoderma harzianum is a potential candidate. These results are in agreement with [7,15,27,34,35].